Hydrodynamic-shear-force mediated interactions between cellular species and their physical environment are central in developmental biology, cancer progression, thrombus formation and other processes. The interactions of the cells with a well-defined surface matrix and with other, cells may be studied under conditions incorporating a bio-rheological environment, as a function of the degree of fluid shear, for example by exposing the cells to biological, chemical or physical shear agonists (or antagonists). Cell-surface and cell to cell interactions may provide an indication of the “activation state” of the cell of interest (essentially, its biochemical and physiological status). The activation state of blood platelets is of particular biomedical interest as the activation state can be an indicator of disease, injury or other physiological stress.
Cardiovascular disease is the leading cause of mortality in Europe and the USA. Cardiovascular events, such as heart attack or stroke, are caused by thrombosis, i.e. the formation of clots which occur as a result of platelet activation. The events initiating thrombosis after vessel injury or plaque rupture are now relatively well understood. At shear flow rates characteristic of arterial circulation, initial platelet adhesion involves Glycoprotein (GP) GPIb-IX-V binding to von Willebrand Factor (vWF), this leads to adherent platelets becoming activated, leading to granule release, functional activation of the platelet-specific integrin GPIIb-IIIa (αIIb-β3) and platelet aggregate formation resulting in an arterial clot or thrombus. This thrombus provides a procoagulant surface leading to the localized generation of thrombin, and hence fibrin, which in part provides structural stability to the developing thrombus.
In contrast to arterial thrombosis, the classic triad described by Virchow in 1856 encapsulates the mechanism of venous thrombosis. This triad of local trauma to the vessel wall, hypercoagulability and stasis leads to venous thrombosis.
The paradigms of arterial and venous thrombosis described above are broadly held. However, while some of the acquired conditions that cause venous thrombosis are understood, there is a remarkable paucity of information on the interaction of platelets with the vessel wall in circulation where many fundamental questions remain to be addressed. For example, the emerging disciplines of genomics and proteomics have identified new proteins in platelets. The roles of these newly identified proteins in thrombosis are not yet understood.
Although thrombosis is prevented in part by aspirin and anti-platelet agents (plavixs and GPIIb IIIa antigonists), heart attacks and stroke still frequently occur. This suggests that the “stickiness” or level of activation of platelets in individuals differs, as does individual response to therapy. In particular, it is difficult to predict those individuals likely to suffer from cardiovascular disease or cardiovascular events.
Unfortunately, platelets are only suited to meaningful assay of their function for approximately four to six hours after being taken from the object. The process of preparing ex vivo platelets shortens this time span. In view of this limitation, typically, only a small number of tests can be performed using prepared platelets. In turn, such studies only provide a small amount of information on platelet function. This means it is yet unclear what constitutes normal platelet function.
The ability to characterise platelet function under haemodynamically relevant conditions would be of benefit, as it would yield valuable prognostic, diagnostic and therapeutic information. In particular, the ability to assess platelet function rapidly in the clinic or at the bedside and more particularly, without any biochemical preparation or alteration of the platelets following their removal from the object, would allow for a more accurate prediction of cardiovascular risk and for anti-platelet therapy to be tailored to the needs of an individual patient in order to minimise the risks of thrombosis and bleeding.
There are a small number of commercially available platelet function analysers on the market. However, these devices have some limitations.
The gold standard test for platelet function is Light Transmission Standard Aggregometry. Unfortunately, the Standard aggregometer test is limited in that the procedure takes a considerable amount of time to perform as the device generally only has a small number of channels (around four channels). Furthermore, aggregometry is not a measure solely or simply of platelet activation status, the aggregation process depending on a complex biochemical and physical sequence of which platelets are one key part.
The PFA (platelet function analyzer)-100™ device measures time to clotting after exposure of whole blood to collagen and epinephrine or collagen and ADP (known as the closure time). However, comparison of the results provided by a PFA-100™ device with those of the gold standard platelet function test, indicates differences and thus the PFA-100 ™ device has limited suitablility.
The Accumetrics Verify Now™ device can be used at the bedside using whole blood to assess platelet function. This device allows the assessment of response to aspirin or Clopidogrel. However, as for the PFA-100™ device, this device only provides a limited amount of information regarding platelet reactivity.
Diamed—Impact-R is a device for testing platelet function under close to physiological conditions. This device tests platelet adhesion and aggregation in anti-coagulated whole blood (for example, using citrate buffer inside blood-draw tubes) under arterial flow conditions (1800 s−1 for 2 min). The Impact-R can be used to study platelet function, screening of primary haemostasis abnormalities and monitoring therapies for treatment of such abnormalities. It can be used for testing both hypo- and hyper-function of platelets and provides a quick method for monitoring the response to various anti-platelet drugs.
Glycotech provides parallel plate flow chambers for research studies under haemodynamic conditions. The chambers currently provided are require assembly to form the complete chamber require the use of a vacuum pump to form a seal of the chamber components; a top plate (or flow deck), silicon rubber gaskets, the dimensions of which, form the flow path area and glass coverslips. For clinical haemodynamic studies, the assembly of the flow chambers and sample provision is technically laborious and requires training. In addition, the volume of blood sample required for haemodynamic studies is considerable. (Using the Glycotech rectangular parallel plate flow chamber, to achieve an arterial flow rate of 1,500 s-1 a blood flow rate of approx. 2.42 ml/min is required). Further, an independent imaging system and operator with expertise in imaging is required. Glycotech is not suitable for point of care use.
Platelet reactivity and the ability of platelets to become activated differs between individuals, and, furthermore, varies within the same individual at different time points. Assessing this variability using the currently available tests is difficult, time inefficient and expensive. Further, whilst a number of platelet function tests are available for clinical application, these tests are less than ideal for point-of-care use. Many of these tests do not account for the potential function of novel or poorly characterised platelet receptors, or use non-physiological agonists to stimulate platelet function and thus are of questionable physiological relevance. Further, they require repeated blood draws, which is not practicable, particularly in infants, as significant volumes of blood are required.
A diagnostic device that uses physiological stimuli on small volumes of blood would be advantageous to provide true point of care evaluation of platelet function for bleeding disorders, thrombotic risk and monitoring drug therapy.